Our overall hypothesis is that PI4KII? (phosphatidylinositol 4 kinase II?), which produces more than 50% of the phosphatidylinositol 4 phosphate (PI4P) in the Golgi, regulates Golgi functions through its localized production of PI4P. The PI4P itself establishes the Golgi's unique organelle identity by recruiting clathrin adapter proteins, such as AP-1, through the coincidence detection of Golgi PI4P and the Arf1 GTPase. In addition, PI4P, as substrate for production of phosphatidylinositol 4, 5 phosphate (PIP2), regulates membrane trafficking from the Golgi. We propose that the localization and enzymatic activity of PI4KII? in the Golgi and Golgi-derived organelles are regulated. Recent data from our lab and others show that the cell has at least two populations of PI4KII? that have different intrinsic catalytic activity and differential association with buoyant vs dense membranes fractions. We recently found that PI4KII?'s catalytic activity, its integral membrane association and partitioning into buoyant noncaveolar "rafts" are critically dependent on its palmitoylation. We therefore propose that these functions of PI4KII? are dynamically regulated by reversible palmitoylation of multiple cysteine residues in PI4KII?. We propose four Specific Aims to test this hypothesis. Aim I. Examine the role of PI4KII? in the generation of dynamic Golgi membrane trafficking carriers that contain PI4KII?. We will characterize these carriers and determine if their generation is dependent on the local synthesis of PI4P per se or downstream synthesis of PIP2. Aim II. Examine the role of PI4P in the recruitment of the AP-3 adaptor protein to endomembranes, to evaluate if it, like the related AP-1, binds target membranes through coincidence detection involving PI4P. Aim III. Determine how palmitoylation regulates PI4KII?. The palmitoylacyl transferase that palmitoylates PI4KII? will be identified and the effect of manipulating its expression on PI4KII? behavior will be examined. Aims I-III focus on the export of membranes from Golgi. Aim IV focuses on the recruitment of PI4KII? to the Golgi. Aim IV. Identify PI4KII?'s primary Golgi targeting motif and interactive proteins. We will identify PI4KII? motifs that are necessary and sufficient to direct PI4KII? to the Golgi prior to palmitoylation and will use an integrated proteomics approach to identify PI4KII? Golgi docking proteins and interactive partners. PUBLIC HEALTH RELEVANCE: Membrane phosphoinositides are major regulators of membrane trafficking, and both their synthesis and degradations are required for dynamic membrane movement and vesicle trafficking within the cell. The experiments in this proposal are designed to examine the role of a lipid kinase that makes an essential lipid in the Golgi. Disruption of this kinase or its misregulation will result in trafficking problems that can contribute to the development of multiple metabolic and neurodegenerative diseases due to improper trafficking of essential components within the cell. Problems with this and other related kinases have already been implicated in neurodegenerative diseases, including Alzheimer's disease.